Characterization of a re‐engineered, mesothelin‐targeted Pseudomonas exotoxin fusion protein for lung cancer therapy

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin‐targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non‐specific toxicity. To overcome both obstacles we developed RG7787, a de‐immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B‐cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin‐treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI‐H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2–3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm3) underwent remission during three treatment cycles with RG7787. Also in two patient‐derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti‐tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti‐tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors

Target binding and thermal stability.

<p>(A) Surface plasmon resonance sensogram of the Fab and CrossFab interacting with their target, hAng2. One out of three runs for the Fab and the CrossFab is shown. Coloured curves represent the measured data at various Fab or CrossFab concentrations, while the result of the global fit to a 1∶1 Langmuir model is illustrated by black curves. The table shows average values and standard deviations derived from triplicate measurements (k_a: association rate; k_d: dissociation rate; K_D: affinity). (B) Measurement of protein stability by temperature-dependent protein autofluorescence emission maximum wavelength.</p

Data collection and refinement statistics.

*<p>Values in parentheses are for the highest-resolution shell. , where is the scaled observed intensity of the <i>j</i>th observation of reflection <i>h</i>, and is the mean value of corresponding symmetry-related reflections.</p

Elbow angles and relative domain orientation.

<p>(A) Superposition of Fab and CrossFab with example structures covering the range of observed elbow angles. Example structures (shown as loops), their orientations and color coding are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Stanfield1" target="_blank">[24]</a>. All structures are superimposed via their V_L domains. The colors of Fab and CrossFab are analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a>. (B) Superposition of the CrossFab with its closest structural homolog (PDB code 3FMG). (C) Relative domain orientation of variable and constant domains in Fab and CrossFab. The pseudo-twofold axes of variable and constant domains are shown as light and dark purple dumbbells, respectively. Molecules are oriented so that the axes connecting the last Fv residues' C-alpha atoms, as well as the Fv pseudo-twofold axis are parallel to the paper plane. An asterisk marks the V_L-C_H1 junction, which is two amino acids longer than the corresponding V_L-C_L junction.</p

CrossMab design.

<p>(A) Schematic representation of the domain crossover leading to CrossFabs. Right: Combination of a Fab and a CrossFab to obtain a CrossMab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone.0061953-Schaefer1" target="_blank">[16]</a>. Antibody domains are symbolized as ovals. Light colors are used for LC domains; darker colors are used for HC domains. This color code is used throughout. (B) Sequence alignment of the elbow and adjacent regions of LC and HC in Fab and CrossFab.</p

Crystal structures and electron density maps.

<p>(A) Comparison of the Fab and CrossFab structures side-by-side. Residues involved in hydrogen bonds between the constant and the variable domains are shown as sticks and hydrogen bonds are shown as red dashed lines. The color code used is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061953#pone-0061953-g001" target="_blank">Figure 1</a>. (B) Superposition of variable domains in Fab and CrossFab. C-alpha atoms excluding CDR H3 were used. Fab and CrossFab variable domains superimpose with an rmsd of 0.43 Å. (C) Superposition of the constant domains in Fab and CrossFab using C-alpha atoms. Fab and CrossFab constant domains superimpose with an rmsd of 0.40 Å. (D) Composite omit maps around the V_HV_L-CH₁C_L interface of the CrossFab and the Fab, contoured at 1.0 sigma using a carve distance of 2.0 Å.</p

ILC Reference Design Report Volume 1 - Executive Summary

The International Linear Collider (ILC) is a 200-500 GeV center-of-mass high-luminosity linear electron-positron collider, based on 1.3 GHz superconducting radio-frequency (SCRF) accelerating cavities. The ILC has a total footprint of about 31 km and is designed for a peak luminosity of 2x10^34 cm^-2s^-1. This report is the Executive Summary (Volume I) of the four volume Reference Design Report. It gives an overview of the physics at the ILC, the accelerator design and value estimate, the detector concepts, and the next steps towards project realization.The International Linear Collider (ILC) is a 200-500 GeV center-of-mass high-luminosity linear electron-positron collider, based on 1.3 GHz superconducting radio-frequency (SCRF) accelerating cavities. The ILC has a total footprint of about 31 km and is designed for a peak luminosity of 2x10^34 cm^-2s^-1. This report is the Executive Summary (Volume I) of the four volume Reference Design Report. It gives an overview of the physics at the ILC, the accelerator design and value estimate, the detector concepts, and the next steps towards project realization